Skip to main content
. 2010 Jan 12;123(3):460–471. doi: 10.1242/jcs.055103

Fig. 5.

Fig. 5.

In vitro reconstitution of clathrin recruitment onto isolated membrane. (A) Subcellular fractionation of dGGA, dCHC and dGM130. The PNS fraction prepared from S2 was separated to membrane (M) and cytosol (C) fractions by ultracentrifugation. Each fraction was subjected to immunoblotting for dCHC and dGGA (upper panel), or dGM130 (lower panel). (B) Isolated membrane was incubated with the cytosol fractions prepared from S2 (lanes 2-6) or from S2 cells depleted of ARF79F (lane 7), plus ATP-regeneration system in the presence or absence of 30 μM of GTP-γS, or with GST or GST-dGGA-VHS-GAT (GST-dGGA-VG) at the indicated concentration. After incubation, membranes in the reaction mixture were precipitated with centrifugation at 20,000 g for 20 minutes and the membrane fractions subjected to immunoblotting with anti-dCHC, anti-dGGA, anti-hARF and anti-dGM130 antibodies. (C) Quantification of the membrane-bound proteins. For each protein, the ratio of the amount in each experimental condition to that in lane 3 was plotted.